Proteases are among the most technologically significant of all enzymes. Among them, proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62), which comprise catalytically active amino acids, also referred to as serine proteases, are particularly important. Acting as nonspecific endopeptidases, they hydrolyze any acid-amide bonds that are located inside peptides or proteins. Their optimum pH is usually in the distinctly alkaline range. An overview of this family of proteases can be found in “Subtilases: Subtilisin-like Proteases,” by R. Siezen, pp. 75-95 in “Subtilisin enzymes,” edited by R. Bolt and C. Betzel, New York, 1996. Subtilases are formed naturally by microorganisms. Among these, the subtilisins formed and secreted by Bacillus species comprise a very significant group within the subtilases.
Proteases, along with other enzymes, are well-established active ingredients present in washing and cleaning products which cause the breakdown of protein-containing stains on the material being cleaned. Ideally, synergistic effects occur between the enzymes and the other constituents of the relevant products. Among the washing and cleaning product proteases, subtilases occupy a prominent position because of their favorable enzymatic properties such as stability or optimum pH. In addition, they are also suitable for a large number of other industrial application possibilities, for example as constituents of cosmetics or in organic chemical synthesis.
The conventional procedure for obtaining new enzymes is to take microorganism-containing samples from natural habitats and culture them in conditions considered to be suitable, (e.g., in an alkaline environment). This yields enriched microorganism cultures that, with a certain probability, also contain enzymes (including alkaline proteases) that are active under the relevant conditions. The microorganisms having the highest-performance enzymes are then selected and purified, (e.g., by plating out onto protein-containing agar plates and measuring the zone of lysis formed). Once isolated, the relevant protease gene is then cloned.
A procedure of this kind is described, for example, in the textbook “Alkalophilic Microorganisms. A New Microbial World,” by K. Horikbshi and T. Akiba (1982), Japan Scientific Societies Press, Springer-Verlag, New York, Heidelberg, Berlin, ISBN 0-387-10924-2, Chapter 2, pp. 9-26. WO 00/24882 A1 also, discloses a method for producing a gene bank that comprises nucleic acids isolated from microorganism-containing samples from different habitats, (e.g., from the rumen), which are cultured under the conditions of interest and thereby enriched. Nucleic acids of interest are then isolated therefrom and cloned.
Alkaline proteases formed naturally, preferably microbially, have already been used in washing and cleaning products. According to Application WO 93/07276 A1, for example, the protease 164-A1 of Chemgen Corp., Gaithersburg, Md., USA, and Vista Chemical Company, Austin, Tex., USA, obtainable from Bacillus spec. 164-A1, is suitable for use in washing and cleaning products. Other examples are the alkaline protease from Bacillus sp. PD138, NCIMB 40338 of Novozymes A/S, Bagsvaerd, Denmark, (WO 93/18140 A1), the proteinase K-16 of Kao Corp., Tokyo, Japan deriving from Bacillus sp. ferm. BP-3376 (U.S. Pat. No. 5,344,770) and, according to WO 96/25489 A1, (Procter & Gamble, Cincinnati, Ohio, USA), the protease from the psychrophilic organism Flavobacterium balustinum. 
Natural proteases are optimized for use in washing and cleaning products, by way of known mutagenesis methods. These include point mutagenesis, deletion, insertion, or fusion with other proteins or protein parts, or alternative modifications. The strategy of introducing deliberate point mutations into a known molecule in order to improve the washing performance of subtilisins is also referred to as “rational protein design.” A similar performance improvement strategy consists of modifying the surface charges and/or the isoelectric point of the molecules, and thereby modifying their interactions with the substrate. Using point mutations, for example, the net charge of the subtilisins can be modified in order to influence substrate binding in particular for use in washing and cleaning products. A further, supplementary strategy consists of increasing the stability of the relevant proteases and thus increasing their effectiveness. Stabilization via coupling to a polymer is described for proteases used in cosmetics, for example, in U.S. Pat. No. 5,230,891 as they are associated with better skin compatibility. For washing and cleaning products in particular, stabilization by way of point mutation introduction is more common.
One modern trend in enzyme development is to combine elements from known related proteins, using statistical methods, to yield new enzymes having properties not hitherto achieved. Such methods are also grouped under the general term “directed evolution.” These include, for example, the following methods: the StEP method (Zhao et al. (1998), Nat. Biotechnol., Vol. 16, pp. 258-261); random priming recombination (Shao et al., (1998), Nucleic Acids Res., Vol. 26, pp. 681-683); DNA shuffling (Stemmer, W. P. C. (1994), Nature, Vol. 370, pp. 389-391); or RACHITT (Coco, W. M. et al. (2001), Nat. Biotechnol., Vol. 19, pp. 354-359). A further shuffling method referred to as a “recombining ligation reaction” (RLR) is described in WO 00/09679 A1.
An overview of the industrially most important alkaline proteases of the subtilisin type will be provided below. Subtilisin BPN′, which derives from Bacillus amyloliquefaciens or B. subtilis, is known from the work of Vasantha et al. (1984) in J. Bacteriol., Volume 159, pp. 811-819, and of J. A. Wells et al. (1983) in Nucleic Acids Research, Volume 11, pp. 7911-7925. Subtilisin BPN′ serves as a reference enzyme for the subtilisins, especially in terms of position numbering.
For example, the position of point mutations in subtilisin described in Application EP 251446 A1, are indicated using the numbering of BPN′ as a reference. Procter & Gamble Corp., of Cincinnati, Ohio, USA, refer to this material as “Protease B.” The BPN′ variants of Application EP 199404 A1 are referred to by Procter & Gamble Corp. as “Protease A.” “A Protease C” is in turn characterized, according to Application WO 91/06637 A1, by further point mutations of BPN′. “Protease D” refers, according to WO 95/10591 A1, to variants of the protease from Bacillus lentus. 
The protease subtilisin Carlsberg is described in the publications of E. L. Smith et al. (1968) in J. Biol. Chem., Volume 243, pp. 2184-2191, and of Jacobs et al. (1985) in Nucl. Acids Res., Volume 13, pp. 8913-8926. It is formed naturally by Bacillus licheniformis, and was and is obtainable under the trade name Maxatase® from Genencor International Inc., Rochester, N.Y., USA, and under the trade name Alcalase® from Novozymes A/S, Bagsvaerd, Denmark.
Protease PB92 is produced naturally by the alkalophilic bacterium Bacillus nov. spec. 92 and is obtainable under the trade name Maxacal® from the Gist-Brocades company, Delft, Netherlands. It is described in: its original sequence in Patent Application EP 283075 A2.
Subtilisins 147 and 309 are marketed under the trade names Esperase® and Savinase®, respectively, by Novozymes. They were originally obtained from Bacillus strains that are disclosed by Application GB 1243784 A.
Subtilisin DY was originally described by Nedkov et al. 1985 in Biol. Chem Hoppe-Seyler, Volume 366, pp. 421-430.
The alkaline protease from B. lentus is an alkaline protease from Bacillus species and is described in Application WO 91/02792 A1. It natively possesses comparatively good stability with respect to oxidation and the action of detergents. Application WO 91/02792 A1 and Patents EP 493398 B1 and U.S. Pat. No. 5,352,604 describe its heterologous expression in the host B. licheniformis ATCC 53926. The claims of the aforesaid US Patent refer to positions 208, 210, 212, 213, and 268 as characteristic of the B. lentus alkaline protease; they correspond, in the numbering of the mature protein, to positions 97, 99, 101, 102, and 157. However this enzyme differs from the mature subtilisin 309 (Savinase®). The three-dimensional structure of this enzyme is described in the publication of Goddette et al. (1992) in J. Mol. Biol., Volume 228, pp. 580-595: “The crystal structure of the Bacillus lentus alkaline protease, Subtilisin BL, at 1.4 Å resolution.” Industrially important variants of this enzyme that are stabilized by point mutagenesis and are suitable in particular for use in washing and cleaning products are disclosed, inter alia, in Applications WO 92/21760 A1, WO 95/23221 A1, WO 02/088340 A2, and WO 03/038082 A2.
Application DE 19857543 A1, for example, discloses liquid to gelled washing and cleaning products having proteases such as those disclosed in WO 95/23221 A1.
The enzyme thermitase, formed naturally by Thermoactinomyces vulgaris, was originally described by Meloun et al. (FEBS Lett. 1983, pp. 195-200). This is a molecule that as a whole exhibits substantial sequence discrepancies compared with the other subtilisins. The homology between the mature thermitase and the alkaline protease proteins from B. lentus DSM 5483 (see below) is not very high, (e.g., 45% identity; 62% similar amino acids).
Proteinase K is also a protease that exhibits comparatively low homology with the alkaline protease from B. lentus: only 33% identity (46% similar amino acids) at the level of the mature proteins. Proteinase K derives originally from the microorganism Tritirachium album Limber, and has been described by K.-D. Jany and B. Mayer (1985) in Biol. Chem. Hoppe-Seyler, Vol. 366, pp. 485-492.
WO 88/07581 A1 discloses proteases TW3 and TW7, which are very similar to one another, for use inter alia in washing and cleaning products.
Bacillopeptidase F from Bacillus subtilis possesses only 30% identity to the B. lentus alkaline protease at the amino-acid level. This enzyme is discussed in the aforementioned work by Siezen et al., but has not hitherto been described or claimed for use in washing and cleaning products.
Application WO 01/68821 A2 describes new subtilisins having good performance with respect to egg stains.
Further alkaline proteases that are formed from microorganisms that can be isolated from natural habitats are described in Applications WO 03/054185 A1 (from Bacillus gibsonii (DSM 14391)), WO 03/056017 A2 (from Bacillus sp. (DSM 14390)), WO 03/055974 A2 (from Bacillus sp. (DSM 14392)), and WO 03/054184 A1 (from Bacillus gibsonii (DSM 14393)). All these Applications also disclose corresponding washing and cleaning products containing these novel alkaline proteases.
Application WO 2004/085639 A1 discloses a serine protease having maximum activity at a pH of 10, from the microorganism Nesterenkonia sp. nov. strain (DSM 15380), together with the gene that codes for it.
A further group of industrially important proteases is represented by the metalloproteases, which require a metal cation as a cofactor. Representatives thereof may also be allocated to the family of the subtilases. Application US 2003/0113895 A1, for example, describes metalloproteases from Gram-positive microorganisms such as B. subtilis, but also from S. cerevisiae, S. pombe, E. coli, and H. influenzae. Washing and cleaning products having metalloproteases are disclosed, for example, by Applications WO 00/60042 A1 and WO 02/36727 A1. In the latter, a specific calcium concentration must be maintained in order to guarantee its activity in the products. Lastly, Application EP 1288282 A1 discloses a mixture of a serine protease and a metalloprotease in dishwashing products.
Further known proteases are the enzymes obtainable under the trade names Durazym®, Relase®, Everlase®, Nafizym, Natalase®, and Kannase® from Novozymes, under the trade names Maxapem®, Purafect®, Purafect OxP®, and Properase® from Genencor, under the trade name Protosol® from Advanced Biochemicals Ltd., Thane, India, and under the trade name Wuxi® from Wuxi Snyder Bioproducts Ltd., China.
As demonstrated by all these efforts performed over a long period of time, a great demand exists for industrially usable proteases that differ—in some cases drastically, in others only at a few positions—from previously known proteases. Such proteases cover a broad spectrum of performance differences, relevant to their use in washing and cleaning products, which in turn represents a large application sector. A suitable protease for washing or cleaning products is thus preferably distinguished by a certain insensitivity to the corresponding conditions (such as the presence of inherently denaturing surfactants, bleaches, high temperatures, etc.), and by good performance with respect to corresponding substrates such as, for example, the proteins present in food residues.
It is equally evident that there still exists an undiminished demand for novel alkaline proteases that are immediately usable per se and that can be further specifically optimized by way of site directed mutagenesis. Such novel proteases are of particular interest in light of the shuffling technologies that have very recently been established. Nucleotide sequences (even if the relevant enzyme itself happens to afford comparatively modest performance) can be shuffled to produce new variants and thus in turn provide entirely new artificial enzymes for use in a variety of industrial applications.